CN115960088A

CN115960088A - Novel coronavirus main protease inhibitor, preparation method and application thereof

Info

Publication number: CN115960088A
Application number: CN202210944223.8A
Authority: CN
Inventors: 杨胜勇; 李琳丽
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2020-07-31
Filing date: 2020-12-25
Publication date: 2023-04-14
Also published as: WO2022021841A1

Abstract

The invention provides a novel inhibitor of coronavirus main protease, a preparation method and application thereof. Specifically provided are compounds represented by formula I, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or optical isomers thereof, or isotopic substitution forms thereof. The compound can effectively inhibit SARS-CoV-2M ^pro Active, can be used for preparing SARS-CoV-2M ^pro Inhibitor for blocking the replication and transcription of SARS-CoV-2 virus in a patient. Preparation of SARS-CoV-2M from the inventive Compounds ^pro The inhibitor, the anti-SARS-CoV-2 medicine and the medicine for preventing and/or treating the novel coronavirus have good application prospects.

Description

Novel inhibitor of coronavirus main protease and preparation method and application thereof

The application is a divisional application provided for an invention patent with the application number of 2020115682827, the application date of which is 2020, 12 and 25.

Technical Field

The invention belongs to the technical field of organic synthetic drugs, and particularly relates to a novel inhibitor of coronavirus main protease, a preparation method and pharmaceutical application thereof.

Background

The genomic RNA of coronaviruses is about 30 ktt long, has a 5 'cap structure and a 3' -poly-a tail, and contains at least 6 Open Reading Frames (ORFs). The first ORF (ORF 1 a/b) occupies about two thirds of the genome length, and translates two polyproteins directly: pp1a and pp1ab, and a-1 frameshift between ORF1a and ORF1 b. These polyproteins consist of a main protease (abbreviated as M) ^pro (ii) a Also known as 3C-like proteases (3 CL) ^pro ) And one or two papain-like proteases (PLPs) to convert into 16 non-structural proteins. These non-structural proteins are involved in the production of subgenomic RNA, encoding four major structural proteins (envelope (E), membrane (M), spinous process (S), and nucleocapsid (N) proteins) and other accessory proteins to complete the viral replication and invasion process.

M ^pro The proteolytic cleavage of the overlapping pp1a and pp1ab into functional proteins is a critical step in the viral replication process. Enzymes essential for viral replication, such as RdRp or nsp13, do not function fully to achieve replication without prior proteolytic release. Thus, inhibition of viral M ^pro Can prevent the generation of infectious virus particles, thereby alleviating the symptoms of the disease.

M ^pro Is conserved among coronaviruses, and M is present in different coronaviruses ^pro Have some common features: the amino acids from N-to C-terminus are numbered in a paired fashion (-P4-P3-P2-P1 ↓ P1'-P2' -P3 '), with the cleavage site between P1 and P1'. In particular, M ^pro There is a unique substrate preference for glutamine at the P1 site (Leu-Gln ↓ (Ser, ala, gly)), which is absent in the host protease, suggesting that by targeting viral M ^pro It is feasible to achieve high selectivity. Thus, the absolute dependence of the virus on the correct function of this protease, coupled with the lack of a homologous human protease, makes M ^pro Becomes an ideal antiviral target.

Therefore, there is a strong need to develop an M that can effectively inhibit SARS-CoV-2 virus ^pro An active drug.

Disclosure of Invention

The invention aims to provide a novel inhibitor of coronavirus main protease, a preparation method and pharmaceutical application thereof.

The invention provides a compound shown as a formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopic substitution form thereof:

/>

wherein X is O or S;

a ring is selected from unsubstituted or substituted by one or more R ₆ Substituted of the following groups: 5-6 membered saturated heterocyclic group, 5-6 membered unsaturated heterocyclic group, saturated hetero condensed ring group, unsaturated hetero condensed ring group; r is ₆ Each independently selected from C _1～6 Alkyl radical, C _1～6 Alkoxy, halogen, hydroxy, cyano, amino, carboxy;

R ₃ is L ₃ M ₀ L ₄ R _3a (ii) a Wherein L is ₃ Selected from the group consisting of _1～4 Alkylene, halogeno C _1～4 Alkylene radical, C _2～4 Alkenylene, halogeno C _2～4 Alkenylene radical, L ₄ Selected from the group consisting of _1～4 Alkylene, halogeno C _1～4 Alkylene radical, M ₀ Selected from among none, O, S, NH, CO, CONH, NHCO, R _3a Is unsubstituted or substituted by one or more R _3b Substituted of the following groups: 5-6 membered aryl, 5-6 membered heteroaryl, unsaturated hetero-condensed ring group, unsaturated condensed ring alkyl; r _3b Each independently is selected from R _3c Substituted or unsubstituted C _1～5 Alkyl radical, by R _3c Substituted or unsubstituted C _1～5 Alkoxy, halogen, by R _3c Substituted or unsubstituted phenyl, NR ₁₄ R ₁₅ Quilt R _3c Substituted or unsubstituted naphthyl, hydroxy; r ₁₄ 、R ₁₅ Each independently selected from hydrogen or C _1～5 Alkyl radical, R _3c Each independently selected from halogen, deuterium, cyano, hydroxyl, amino, carboxyl;

R ₄ selected from the following groups unsubstituted or substituted with one or more substituents: 5-6 membered aryl, 5-6 membered heteroaryl, C _1～5 Alkyl, COOR ₁₀ (ii) a The substituents are each independently selected from = O, hydroxy, nitro, amino, carboxy, halogen, C _1～5 An alkyl group; r is ₁₀ Is C _1～5 An alkyl group;

R ₅ is selected from COR ₈ Or WCOOR ₇ (ii) a Wherein R is ₈ Selected from hydrogen or

W is selected from the group consisting of _1～4 Alkylene radical, C _2～4 Alkenylene radical, C _2～4 Alkynylene, R ₇ Is selected from C _1～6 An alkyl group; m is selected from among nothing, CO, NH, CONH, NHCO, COO or OCO, L ₀ Selected from the group consisting of _1～4 Alkylene radical, C _2～4 Alkenylene radical, L ₁ Selected from among none, C _1～4 Alkylene radical, C _2～4 Alkenylene radical, R ^8a Is selected from C _1～5 Alkyl, halogenated C _1～5 Alkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclyl, 5-6 membered aryl or 5-6 membered heteroaryl.

Further, the structure of the compound is shown as formula II, formula III or formula IV:

wherein X is O or S;

n is selected from an integer of 0 to 3, preferably an integer of 0 to 2;

R ₁ 、R ₂ each independently selected from hydrogen, C _1～5 Alkyl radical, C _1～5 Alkoxy, halogen, hydroxy, cyano, amino, carboxy;

R ₃ is L ₃ M ₀ L ₄ R _3a (ii) a Wherein L is ₃ Selected from the group consisting of _1～4 Alkylene, halogeno C _1～4 Alkylene radical, C _2～3 Alkenylene radical, L ₄ Selected from among none, C _1～4 Alkylene, halogeno C _1～4 Alkylene radical, M ₀ Selected from among none, O, S, NH, CO, CONH, NHCO, R _3a Is unsubstituted or substituted by one or more R _3b Substituted of the following groups: phenyl, phenyl,

R _3b Each independently selected from C _1～4 Alkyl, halogen substituted C _1～4 Alkyl, deuterated C _1～4 Alkyl, cyano-substituted C _1～4 Alkyl radical, C _1～4 Alkoxy, halogen substituted C _1～4 Alkoxy, deuterated C _1～4 Alkoxy, cyano-substituted C _1～4 Alkoxy, halogen, phenyl, halogenated phenyl, NR ₁₄ R ₁₅ 、/>

Hydroxy radical, R ₁₄ 、R ₁₅ Each independently selected from hydrogen or C _1～4 An alkyl group;

R ₈ selected from hydrogen or

M is selected from,CO, NH, CONH, NHCO, COO or OCO, L ₀ Selected from the group consisting of _1～3 Alkylene radical, C _2～4 Alkenylene radical, L ₁ Selected from among none, C _1～3 Alkylene radical, C _2～4 Alkenylene radical, R ^8a Is selected from C _1～4 Alkyl, halogenated C _1～4 Alkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclyl, 5-6 membered aryl or 5-6 membered heteroaryl.

Further, R ₁ 、R ₂ Each independently selected from hydrogen, C _1～4 Alkyl radical, C _1～4 Alkoxy, halogen, hydroxy;

R ₃ is selected from

L ₃ M ₀ L ₄ R _3a ；L ₃ Selected from the group consisting of _1～3 Alkylene, halogeno C _1～3 Alkylene radical, C _2～3 Alkenylene radical, L ₄ Selected from the group consisting of _1～3 Alkylene, halogeno C _1～3 Alkylene, M ₀ Selected from among O, NH, CO, CONH, R _3a Is phenyl, substituted by one or more R _3b Substituted phenyl radicals, R _3b Each independently selected from C _1～4 Alkyl, halogen substituted C _1～4 Alkyl, deuterated C _1～4 Alkyl, cyano-substituted C _1～4 Alkyl radical, C _1～4 Alkoxy, halogen substituted C _1～4 Alkoxy, deuterated C _1～4 Alkoxy, cyano-substituted C _1～4 Alkoxy, halogen, phenyl, halogenated phenyl, NR ₁₄ R ₁₅ 、/>

Hydroxy, R ₁₄ 、R ₁₅ Each independently selected from hydrogen or C _1～3 An alkyl group;

R ₄ is selected from

C _1～2 Alkyl, COOR ₁₀ Substituted or unsubstituted phenyl; the substituent is selected from hydroxyl and nitro; r _a1 、R _a2 Each independently selected from hydrogen, C _1～3 Alkyl, halogen; r ₁₀ Is C _1～3 An alkyl group;

R ₈ selected from hydrogen, CONHR ₁₁ 、L ₂ COOR ₁₂ 、C _1～4 Alkyl, halogenated C _1～4 An alkyl group; r is ₁₁ Selected from 3-to 6-membered saturated cycloalkyl, C _1～4 Alkyl, benzyl, or the like,

L ₂ Is C _1～2 Alkylene radical, C _2～3 Alkenylene radical, R ₁₂ Is C _1～3 An alkyl group.

Further, the formula II is shown as a formula II-1 or a formula II-2:

wherein X is O or S, preferably O;

R ₁ 、R ₂ each independently selected from hydrogen, C _1～3 Alkyl, preferably methyl;

m is an integer of 0 to 3, R _3b Each independently selected from phenyl, halogenated phenyl, halogen, C _1～3 Alkyl, halo or deuterated C _1～3 Alkyl radical, C _1～3 Alkoxy, halo or deuterated C _1～3 Alkoxy, hydroxy;

R _a1 、R _a2 each independently selected from hydrogen, C _1～3 Alkyl, halogen;

R _b selected from hydrogen, C _1～3 Alkyl, halogenated C _1～3 An alkyl group;

L ₃ selected from among none, C _1～2 Alkylene, halogeno C _1～2 Alkylene radical, C ₂ Alkenylene radical, L ₄ Selected from among none, C _1～3 Alkylene, halogeno C _1～3 Alkylene radical, M ₀ Selected from none, O, NH, CO, CONH;

the halogen is preferably chlorine or fluorine.

Further, the structure of the compound is one of the following structures:

/>

/>

/>

/>

/>

the invention also provides a pharmaceutical composition, which is a preparation prepared by taking the compound, or pharmaceutically acceptable salt, or stereoisomer, or optical isomer, or isotope substitution form thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

The invention also provides the use of the above compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopic substitution form thereof, for the preparation of inhibitors of coronavirus proteolytic enzyme; preferably, the coronavirus proteolytic enzyme is coronavirus main protease; more preferably, the coronavirus proteolytic enzyme is SARS-COV-2M ^pro 。

The invention also provides the application of the compound or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the optical isomer thereof, or the isotope substitution form thereof in preparing the anti-coronavirus medicament, preferably, the coronavirus is novel coronavirus SARS-CoV-2.

The invention also provides the application of the compound, or the pharmaceutically acceptable salt, the stereoisomer, the optical isomer or the isotopic substitution form thereof in the preparation of medicines for preventing and/or treating SARS-COV-2M ^pro Use in medicine of related diseases, preferably, the SARS-COV-2M ^pro The related disease is a novel coronavirus COVID-19.

Furthermore, the coronavirus proteolytic enzyme inhibitor, the anti-coronavirus drug or the drug for preventing and/or treating viral pneumonia can inhibit SARS-COV-2M ^pro And/or can inhibit SARS-COV-2 infection of cells.

Definitions of terms used in connection with the present invention: unless otherwise indicated, the initial definitions provided for by a group or term herein apply to that group or term throughout the specification; for terms not specifically defined herein, the meanings that would be given to them by a person skilled in the art are to be given in light of the disclosure and the context.

The minimum and maximum values of the content of carbon atoms in hydrocarbon groups are indicated by a prefix, e.g. prefix C _a～b Alkyl represents any alkyl group containing from "a" to "b" carbon atoms. E.g. C _1～6 Alkyl refers to a straight or branched chain alkyl group containing 1 to 6 carbon atoms.

By "substituted" herein is meant that 1, 2 or more hydrogen atoms in the molecule are replaced by other different atoms or molecules, including 1, 2 or more substitutions on the same or different atoms in the molecule.

"isotopically substituted forms" refer to compounds wherein one or more than two atoms are replaced by their corresponding isotopes, for example compounds wherein hydrogen is replaced by protium, deuterium or tritium.

By "pharmaceutically acceptable" is meant a carrier, cargo, diluent, excipient, and/or salt that is generally chemically or physically compatible with the other ingredients that make up the pharmaceutical dosage form, and with the recipient.

"salts" are acid and/or base salts of a compound or a stereoisomer thereof with inorganic and/or organic acids and/or bases, and also include zwitterionic (inner) salts, as well as quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. Or by mixing the compound, or a stereoisomer thereof, with a certain amount of an acid or a base, as appropriate (e.g., an equivalent amount). These salts may form precipitates in the solution which are collected by filtration, or they may be recovered by evaporation of the solvent, or they may be prepared by reaction in an aqueous medium followed by lyophilization.

The "pharmaceutically acceptable salt" may be a hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate salt of the compound.

Halogen is fluorine, chlorine, bromine or iodine.

"aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., rings which share adjacent pairs of carbon atoms) group having a conjugated pi-electron system, such as phenyl. The aryl group does not contain heteroatoms such as nitrogen, oxygen or sulfur, and the point of attachment to the parent must be at a carbon atom in the ring having a conjugated pi-electron system. The aryl group may be substituted or unsubstituted. "5-to 6-membered aryl" means an aryl group having 5 or 6 carbon atoms in the ring.

"heteroaryl" refers to a heteroaromatic group containing one to more heteroatoms. Heteroatoms as referred to herein include oxygen, sulfur and nitrogen. Such as furyl, thienyl, pyridyl, pyrazolyl, and the like. The heteroaryl group may be optionally substituted or unsubstituted. "5-to 6-membered heteroaryl" refers to heteroaryl having 5 or 6 ring atoms.

"cycloalkyl" refers to a saturated or unsaturated cyclic hydrocarbon substituent; the cyclic hydrocarbon may be monocyclic or polycyclic. For example, "3-to 6-membered saturated cycloalkyl" refers to a saturated cycloalkyl group having 3 to 6 carbon atoms in the ring.

"heterocyclyl" refers to a saturated or unsaturated cyclic hydrocarbon substituent; the cyclic hydrocarbon may be monocyclic or polycyclic and carries at least one ring heteroatom (including but not limited to O, S or N). For example, "3-to 6-membered saturated heterocyclic group" means a saturated heterocyclic group having 3 to 6 ring atoms.

"fused cycloalkyl" refers to a polycyclic cycloalkyl group in which two rings share two adjacent carbon atoms.

"Heterofused cyclic" refers to polycyclic heterocyclic groups containing at least 1 heteroatom, and wherein two rings in the polycyclic heterocyclic group share two adjacent carbon or heteroatoms.

"alkylene" refers to a group in which one atom of an alkyl group has been removed. Such as C ₁ Alkylene group:

C ₂ alkylene group:

"alkenylene" refers to a group resulting from the loss of one atom from an alkenyl group. E.g. C ₂ Alkenyl:

"alkynylene" refers to a group that has one atom of an alkynyl group removed. E.g. C ₂ Alkynyl:

the experimental result shows that the invention provides a new type coronavirus main protease M which can be effectively inhibited ^pro An active compound which can effectively inhibit the replication of SARS-COV-2 virus in cells, inhibit the infection of SARS-COV-2 in cells and resist the infection of SARS-COV-2 in vivo of transgenic mice; reducing virus load of lung of transgenic mouse infected by SARS-COV-2, reducing gene expression level of chemokine ligand 10 (CXCL 10) and beta-type interferon (IFN-beta) in mouse lung, reducing quantity of neutrophilic granulocyte (NEU) and Macrophage (MAC) in mouse lung, and improving pathological injury of mouse lung. Also, the invention provides compoundsThe product also has good in vivo safety and pharmacokinetic properties. Preparation of SARS-CoV-2M from the inventive Compounds ^pro The inhibitor, the medicine for resisting SARS-CoV-2 and the medicine for preventing and/or treating the novel coronavirus have very good application prospects.

It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and common practice in the field.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows that compound 26 is against SARS-COV-2M ^pro Inhibitory activity curve of (1).

FIG. 2 shows that compound 33 is against SARS-COV-2M ^pro Inhibitory activity curve of (1).

FIG. 3 shows that compound 37 is against SARS-COV-2M ^pro Inhibitory activity curve of (1).

FIG. 4 shows the inhibition of SARS-COV-2 replication in human alveolar epithelial cells by the compound.

FIG. 5 pulmonary viral load of SARS-CoV-2 infected mice.

FIG. 6 Lung pathological tissue section (3 dpi) of SARS-CoV-2 infected mouse.

FIG. 7 lung representative cytokine expression levels (3 dpi) of SARS-CoV-2 infected mice.

FIG. 8 Lung neutrophil and macrophage counts (3 dpi) of SARS-CoV-2 infected mice.

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

Example 1: preparation of Compound 1

Compound 1 of the present invention is prepared according to the above preparation route, wherein the reaction conditions of the steps are as follows:

i. a, 2-fluoro-malonic acid dimethyl ester, benzyl alcohol, toluene, p-toluenesulfonic acid, 110 ℃; b, isopropanol, n-hexane, -10 ℃;

ii. Isopropanol, sodium hydroxide, water, 45 ℃;

iii, anhydrous tetrahydrofuran, isopropyl magnesium chloride tetrahydrofuran solution, ar,0 ℃;

iv, anhydrous tetrahydrofuran, N' -carbonyldiimidazole, ar,0 ℃;

v, ethyl acetate, 10% palladium on carbon, hydrogen, room temperature;

vi, dichloromethane and dioxane hydrochloride solution;

vii, dichloromethane, N, N, N ', N' -tetramethyl-O- (7-azabenzotriazol-1-yl) hexafluorophosphate urea, N, N-diisopropylethylamine, -20 ℃;

viii, dichloromethane, trifluoroacetic acid;

ix, dichloromethane, N' -tetramethyl-O- (7-azabenzotriazol-1-yl) urea hexafluorophosphate, N-diisopropylethylamine-20 ℃;

the specific synthesis steps are as follows:

intermediate 2: preparation of dibenzyl 2-fluoromalonate

Dimethyl 2-fluoropropanoate (10g, 66.6mmol, 1.0eq) and benzyl alcohol (35mL, 338.2mmol, 5.0eq) were dissolved in 100mL of toluene, 1.15g of p-toluenesulfonic acid (6.7mmol, 0.1eq) was added, the reaction was refluxed, and TLC was used to monitor the reaction, after about 8 hours, the reaction was completed. Cooling to room temperature, evaporating toluene under reduced pressure, adding 15mL of isopropanol, stirring uniformly, slowly adding 30mL of n-hexane under stirring, placing in a-10 ℃ cold trap, and continuously stirring for 2 hours to precipitate a large amount of white solid. Suction filtration is carried out, the filter cake is washed twice by 10mL multiplied by 2 frozen n-hexane, and the filter cake is dried under vacuum and reduced pressure at 30 ℃ to obtain 18.4g of product with the yield of 91.4 percent. ¹ H NMR(400MHz,DMSO-d6)δ7.64–6.91(m,10H),6.00(d,J＝46.3Hz,1H),5.30–5.20(m,4H).

Intermediate 3: preparation of 2-fluoro-malonic acid monobenzyl ester

Dibenzyl 2-fluoropropanedioate (18.4g, 60.9mmol, 1.0eq) was dissolved in 100mL of isopropanol, the temperature was raised to 45 ℃ and sodium hydroxide (2.55g, 63.9mmol, 1.05eq) was dissolved in 60mL of water and then the solution was slowly dropped for > 1 hour. After the dropwise addition, the reaction was continued for 30 minutes, isopropanol was distilled off under reduced pressure, 50mL of water was added, and the pH was adjusted to about 9 with a saturated sodium bicarbonate solution. The aqueous phase is extracted twice with dichloromethane 20mL × 2, the pH of the aqueous phase is adjusted to 1-2 with 6mol/L hydrochloric acid, extracted three times with 40mL × 3 isopropyl ether, the organic phases are combined and washed once with 30mL saturated brine. Adding anhydrous magnesium sulfate into the organic phase, drying, filtering, concentrating to obtain viscous residue, adding 60mL of n-hexane, stirring overnight, separating out white solid, filtering, and vacuum drying the filter cake at 40 ℃ under reduced pressure to obtain 6.5g of product with the yield of 50.3%. ¹ H NMR(400MHz,Chloroform-d)δ7.41–7.32(m,5H),5.87(s,2H),5.39(d,J＝47.9Hz,1H),5.31(s,1H).

Preparation of intermediate 4

2-Fluoromalonic acid monobenzyl ester is dissolved in anhydrous tetrahydrofuran (2 mL/mmol), replaced by argon for protection, cooled to 0 ℃, and slowly added with isopropyl magnesium chloride tetrahydrofuran solution (2M tetrahydrofuran solution, 2.0 eq) to obtain white suspension. Stirring is continued for 1 hour at 0 ℃, and the product suspension is directly used for the next reaction.

Intermediate 6: preparation of 1-benzyl 6-methyl (4S) -4- (((tert-butoxycarbonyl) amino) -2-fluoro-3-oxoadipate

Boc-L-aspartic acid 4-methyl ester (2.2g, 8.8mmol, 1.0eq) was dissolved in 50mL of anhydrous tetrahydrofuran, replaced with argon for protection, cooled to 0 ℃, CDI (1.5g, 9.3mmol, 1.05eq) was added, and the reaction was allowed to proceed for 1 hour with incubation. The reaction solution is cooled to-20 ℃,1.5 eq of the intermediate 4 is slowly added, the reaction is kept for 1 hour, and then the temperature is raised to room temperature for reaction for 6 hours. Slowly pouring the reaction solution into 300mL of 2M dilute hydrochloric acid in an ice water bath, extracting with 100mL of multiplied by 3 ethyl acetate for three times, combining organic phases, washing with saturated sodium bicarbonate solution until the organic phases are alkalescent, washing with 50mL of saturated saline water once, adding anhydrous magnesium sulfate for drying, filtering and concentrating, and directly using the obtained crude product for the next reaction.

Intermediate 7: preparation of methyl (S) -3- (((tert-butoxycarbonyl) amino) methyl-5-fluoro-4-oxopentanoate

Adding 50mL of ethyl acetate into the crude intermediate 6 obtained in the previous step, adding 200mg of 10% palladium carbon, performing hydrogen replacement, reacting at room temperature under hydrogen overnight, filtering, concentrating, and reacting the obtained crude product with petroleum ether: ethyl acetate =10: 1.5g of colorless oil was obtained by mobile phase column chromatography in 65% yield. ¹ H NMR(400MHz,Chloroform-d)δ5.51(d,J＝8.0Hz,1H),5.28–5.06(m,2H),4.73–4.52(m,1H),3.70(s,3H),3.08(dd,J＝17.2,4.6Hz,1H),2.84(dd,J＝17.2,5.0Hz,1H),1.46(s,9H).

Intermediate 8: preparation of (S) -3-amino-5-fluoro-4-oxopentanoic acid methyl ester

500mg of intermediate 7 was dissolved in 5mL of dichloromethane, then 5mL of dioxane hydrochloride was added, and after completion of the reaction, spin-dried to give intermediate 8 in 91.2% yield. ¹ H NMR(400MHz,Chloroform-d)δ5.25-5.10(m,2H),4.53(dd,J＝8.7,1.0Hz,2H),4.44(d,J＝7.9Hz,1H),3.69(s,3H),2.83–2.71(m,2H).

Intermediate 11: preparation of methyl (1H-indole-2-carbonyl) -L-proline

1H-indole-2-carboxylic acid (1g, 6.21mmol, 1.0eq) was dissolved in methylene chloride, HATU (2.81g, 7.40mmol, 1.2eq) was added at-20 ℃ followed by L-proline methyl ester hydrochloride (1.03g, 6.21mmol, 1.0eq) and finally DIEA (3mL, 18.51mmol, 3.0eq) was added, and the reaction was monitored by TLC. After the reaction was complete, extraction was performed with aqueous solution and DCM, and the organic layer was concentrated and isolated by column chromatography to give intermediate 11 (1.53 g) in 75.2% yield. ¹ H NMR(400MHz,DMSO-d ₆ )δ7.68(dt,J＝7.4,1.5Hz,1H),7.43(dd,J＝7.4,1.6Hz,1H),7.26(td,J＝7.5,1.7Hz,1H),7.19–7.14(m,2H),4.31(t,J＝7.0Hz,1H),3.72(td,J＝7.1,2.3Hz,2H),3.68(s,3H),2.11–2.00(m,2H),1.93–1.81(m,2H)。

Intermediate 12: preparation of (1H-indole-2-carbonyl) -L-proline

500mg of intermediate 11 was dissolved in10 mL of dichloromethane, then 5mL of trifluoroacetic acid was added and after the reaction was complete, the intermediate 12 was spun dry to yield 378mg, which was used directly as the first reaction. The yield thereof was found to be 91.2%.

Compound 1: preparation of methyl (S) -3- ((S) -1- (1H-indole-2-carbonyl) pyrrolidine-2-carboxamide) -5-fluoro-4-oxopentanoic acid

Intermediate 12 (168mg, 0.61mmol, 1.0eq) was dissolved in dichloromethane, HATU (280mg, 0.73mmol, 1.2eq) was added at-20 ℃ followed by intermediate 8 (100mg, 0.61mmol, 1.0eq) and finally DIEA (301. Mu.L, 1.83mmol, 3.0eq) was added, and the reaction was monitored by TLC. After the reaction, the mixture was extracted with an aqueous solution and DCM, the organic layer was concentrated and separated by column chromatography to give compound 1 with a yield of 34%. ¹ H NMR(400MHz,DMSO)δ11.55(s,1H),8.69(s,1H),7.65(d,J＝7.6Hz,1H),7.46(d,J＝8.3Hz,1H),7.20(m,1H),7.06(d,J＝7.8Hz,2H),5.26(m,2H),4.60(m,1H),4.49(m,1H),3.96(dd,J＝15.0,7.4Hz,2H),3.61(s,3H),2.86(m,1H),2.60(dd,J＝15.9,7.7Hz,1H),2.02(m,2H),1.82(m,2H).HRMS m/z(ESI)calcd for C ₂₀ H ₂₅ FN ₄ O ₅ [M+H] ⁺ 403.1543found:404.1476。

Example 2: preparation of Compound 3

Compound 3 of the present invention is prepared according to the above preparation route, wherein the reaction conditions of the steps in the route are as follows:

i. Boc-L-glutamic acid dimethyl ester, liHMDS tetrahydrofuran solution, argon, anhydrous tetrahydrofuran, -78 ℃;

ii. (2s, 4r) -dimethyl 2- (tert-butoxycarbonylamino) -4- (cyanomethyl) glutarate, anhydrous methanol, cobalt chloride hexahydrate, sodium borohydride;

iii, (S) -methyl 2- (tert-butoxycarbonylamino) -3- ((S) -2-carbonylpyrrolidin-3-yl) propionate, lithium hydroxide monohydrate, tetrahydrofuran, 0 ℃;

iv, anhydrous tetrahydrofuran, ar, N' -carbonyldiimidazole, 0 ℃;

v, ethyl acetate, 10% palladium on carbon, hydrogen, room temperature;

vi, dichloromethane and dioxane hydrochloride solution;

vii, dichloromethane, N' -tetramethyl-O- (7-azabenzotriazol-1-yl) urea hexafluorophosphate, N-diisopropylethylamine, -20 ℃;

viii, dichloromethane, trifluoroacetic acid;

the specific synthesis steps are as follows:

intermediate 14: preparation of (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (cyanomethyl) glutaric acid dimethyl ester

Boc-L-glutamic acid dimethyl ester (12g, 43.6mmol, 1.0eq) was dissolved in 100mL of anhydrous tetrahydrofuran, replaced with argon for protection, cooled to-78 ℃,94 mL of LiHMDS tetrahydrofuran solution (1M tetrahydrofuran solution, 94mmol, 2.2eq) was slowly added dropwise, and the reaction was allowed to proceed for 1 hour after the addition was completed. 3.24mL bromoacetonitrile (46.6 mmol, 1.1eq) was added slowly dropwise to the reaction mixture, the reaction was incubated for 6 hours and quenched with 50mL saturated ammonium chloride solution. The quenched reaction was warmed to room temperature, extracted three times with 60mL × 3 ethyl acetate, the organic phases were combined, washed with 50mL saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by petroleum ether: ethyl acetate =4:1 mobile phase column chromatography gave 9.36g of pale yellow oil, yield 68.3%. ¹ H NMR(400MHz,Chloroform-d)δ5.11(d,J＝8.6Hz,1H),4.39(s,1H),3.77(s,3H),3.76(s,3H),2.90–2.82(m,1H),2.82–2.74(m,2H),2.28–2.06(m,2H),1.45(s,9H).

Intermediate 15: preparation of methyl (S) -2- (((tert-butoxycarbonyl) amino) methyl-3- ((S) -2-oxopyrrolidin-3-yl) propionate

(2S, 4R) -dimethyl-2- (tert-butoxycarbonylamino) -4- (cyanomethyl) glutarate (9.36g, 29.8mmol, 1.0eq) was dissolved in 150mL of anhydrous methanol and cooled to 0 ℃. Cobalt chloride hexahydrate (4.25g, 18mmol, 0.6eq) was added, sodium borohydride (6.76g, 180mmol, 6.0eq) was added in portions, and after the addition, the temperature was raised to room temperature, the reaction was allowed to proceed overnight, and the completion of the reaction was monitored by TLC. Adding 50mL saturated ammonium chloride solution to quench reaction, evaporating methanol under reduced pressure, extracting with 100mL × 3 ethyl acetate three times, combining organic phases, washing with 200mL × 3 saturated ammonium chloride solution three times, washing with 200mL × 3 saturated saline three times, adding anhydrous magnesium sulfate to the organic phase, drying, filtering, concentrating to obtain crude productUsing petroleum ether: ethyl acetate =1:1 mobile phase column chromatography gave 3.94g of white solid with a yield of 46.2%. ¹ H NMR(400MHz,Chloroform-d)δ5.92(s,1H),5.49(d,J＝8.4Hz,1H),4.41–4.26(m,1H),3.74(s,3H),3.45–3.26(m,2H),2.58–2.39(m,2H),2.27–2.07(m,1H),1.98–1.78(m,2H),1.44(s,9H).

Intermediate 16: preparation of (S) -2- ((tert-butoxycarbonyl) amino) -3- ((S) -2-oxopyrrolidin-3-yl) propionic acid

(S) -methyl 2- (tert-butoxycarbonylamino) -3- ((S) -2-carbonylpyrrolidin-3-yl) propionate (0.88g, 3.1mmol, 1.0eq) was dissolved in10 mL tetrahydrofuran and cooled to 0 ℃. Lithium hydroxide monohydrate (0.64g, 15.4mmol, 5.0eq) was dissolved in10 mL of water and then slowly dropped, and after dropping, the reaction was kept for 4 hours, and the completion of the reaction was monitored by TLC. Adjusting the pH value of saturated citric acid aqueous solution to be neutral, evaporating tetrahydrofuran under reduced pressure, extracting once by 10mL ethyl acetate, adjusting the pH value of an aqueous phase to be 3-4 by using the saturated citric acid aqueous solution, extracting three times by 20mL multiplied by 3 ethyl acetate, combining organic phases, washing by using 20mL saturated saline, adding anhydrous magnesium sulfate, drying, filtering and concentrating to obtain 0.78g of off-white solid with the yield of 93.2%. ¹ H NMR(400MHz,Chloroform-d)δ7.19(s,1H),5.69(d,J＝7.9Hz,1H),4.35(q,J＝7.6Hz,1H),3.48–3.31(m,2H),2.70–2.55(m,1H),2.51–2.36(m,1H),2.27–2.12(m,1H),1.99–1.80(m,2H),1.44(s,9H).

Intermediate 17: preparation of benzyl (4S) -4- ((tert-butoxycarbonyl) amino) -2-fluoro-3-oxo-5- ((S) -2-oxopyrrolidin-3-yl) pentanoate

(S) -2- ((tert-butoxycarbonyl) amino) -3- ((S) -2-carbonylpyrrolidin-3-yl) propionic acid (2.4g, 8.8mmol, 1.0eq) was dissolved in 50mL of anhydrous tetrahydrofuran, purged with argon, cooled to 0 ℃ and reacted with CDI (1.5g, 9.3mmol, 1.05eq) under heat for 1 hour. The reaction solution is cooled to-20 ℃,1.5 eq of the intermediate 4 is slowly added, the reaction is kept for 1 hour, and then the temperature is raised to room temperature for reaction for 6 hours. Slowly pouring the reaction solution into 300mL of 2M diluted hydrochloric acid in an ice water bath, extracting with 100mL of multiplied by 3 ethyl acetate for three times, combining organic phases, washing with saturated sodium bicarbonate solution to be alkalescent, washing with 50mL of saturated saline once, adding anhydrous magnesium sulfate for drying, filtering, concentrating, and directly using the obtained crude product for the next reaction.

Intermediate 18: preparation of tert-butyl ((S) -4-fluoro-3-oxo-1- ((S) -2-oxopyrrolidin-3-yl) butan-2-yl) carbamate

Adding 50mL of ethyl acetate into the crude intermediate 17 obtained in the previous step, adding 200mg of 10% palladium carbon, replacing with hydrogen, reacting overnight at room temperature under hydrogen, filtering, concentrating, and reacting the obtained crude product with petroleum ether: ethyl acetate =1:1 mobile phase column chromatography gave 1.3g of white solid with a yield of 50%. ¹ H NMR(400MHz,Chloroform-d)δ5.99(d,J＝7.5Hz,1H),5.91(s,1H),5.31–4.95(m,2H),4.56(s,1H),3.42–3.32(m,2H),2.56–2.42(m,2H),2.10–1.97(m,1H),1.96–1.81(m,2H),1.45(s,9H).

Intermediate 19: preparation of (S) -3- ((S) -2-amino-4-fluoro-3-oxobutyl) pyrrolidin-2-one

500mg of intermediate 18 was dissolved in 5mL of dichloromethane, then 5mL of dioxane hydrochloride was added, and after completion of the reaction, the intermediate 19 was obtained by spin-drying, the yield was 85%.

Intermediate 22: preparation of Ethyl (1S, 3aR,6 aS) -2- (2, 4-dichlorophenoxy) acetyl) octahydrocyclopenta [ c ] pyrrole-1-carboxylate.

2, 4-Dichlorophenoxyacetic acid (intermediate 21,0.58g, 2.62mmol), 2- (7-benzotriazol oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (1.2g, 3.14mmol), N, N-diisopropylethylamine (1.3mL, 7.86mmol), and (1S, 3aR,6 aS) -octahydrocyclopenta [ c ] ester]Pyrrole-1-carboxylic acid ethyl ester hydrochloride (intermediate 20,0.58g, 2.62mmol) was dissolved in 15mL of ultra dry N, N-dimethylformamide and reacted at 25 ℃ under argon protection for 12 hours, d reaction added 4 times volume of water, extracted three times with dichloromethane, combined organic phases washed with saturated ammonium chloride solution, saturated sodium carbonate solution, dried over anhydrous sodium sulfate and filtered, silica gel stirred column chromatography (petroleum ether/ethyl acetate = 1). ¹ H NMR(400MHz,MeOD)δ7.42(d,J＝5.4Hz,1H),7.26–7.19(m,1H),6.97(d,J＝8.9Hz,1H),4.80-7.72(m,2H),4.28(d,J＝3.6Hz,1H),4.22–4.10(m,2H),3.87(d,J＝10.6Hz,1H),3.63–3.48(m,1H),3.57(d,J＝10.5Hz,2H),2.71–2.61(m,1H),2.08–1.84(m,1H),1.83–1.46(m,4H),1.31–1.17(m,3H).ESI-MS(m/z):386.02(M+H) ⁺ .

Intermediate 23: preparation of (1S, 3aR, 6aS) -2- (2, 4-dichlorophenoxy) acetyl) octahydro-cyclopenta [ c ] pyrrole-1-carboxylic acid

Ethyl (1s, 3ar,6 as) -2- (2, 4-dichlorophenoxy) acetyl) octahydrocyclopenta [ c ] pyrrole-1-carboxylate (intermediate 22, 200mg, 0.52mmol) was dissolved in 20mL of methanol, then 2M sodium hydroxide solution (10 mL) was added and the reaction stirred at 25 ℃ for 4 hours, after TLC monitoring the reaction was completed, methanol was spun off, the pH was adjusted to weak acidity with hydrochloric acid, dichloromethane was extracted three times, and the combined organic phases were dried over anhydrous sodium sulfate and then selected to dry to obtain a crude product which was directly subjected to the next reaction.

Compound 3: preparation of (1S, 3aR, 6aS) -2- (2, 4-dichloro) acetyl) -N- ((S) -4-fluoro-3-oxo-1- ((S) -2-oxo-3-yl) butan-2-yl) octahydrocyclopenta [ c ] pyrrole-1-carboxamide

Intermediate 23 (168mg, 0.61mmol, 1.0eq) was dissolved in dichloromethane, HATU (280mg, 0.73mmol, 1.2eq) was added at-20 ℃ followed by intermediate 19 (100mg, 0.61mmol, 1.0eq) and finally DIEA (301. Mu.L, 1.83mmol, 3.0eq) was added, and the reaction was monitored by TLC. After the reaction is finished, extracting with an aqueous solution and DCM, concentrating an organic layer, and separating by column chromatography to obtain a compound 3 with the yield of 34%. ¹ H NMR(400MHz,DMSO)δ8.61(d,J＝7.4Hz,2H),8.29(d,J＝7.7Hz,1H),8.15(d,J＝8.5Hz,1H),7.65(s,1H),7.56(d,J＝7.0Hz,2H),7.41(td,J＝11.1,6.0Hz,4H),6.75(dd,J＝15.9,6.1Hz,1H),5.15(m,2H),4.39(s,1H),3.62(d,J＝4.1Hz,2H),3.16(m,1H),3.11(m,2H),2.28(d,J＝36.4Hz,1H),2.12(s,1H),1.96(m,1H),1.62(m,2H),1.50(dd,J＝15.5,8.6Hz,2H),0.88(m,6H).HRMS m/z(ESI)calcd for C ₂₃ H ₃₀ FN ₃ O ₄ [M+H] ⁺ 432.2293found:432.2291。

Example 3: preparation of Compound 9

Compound 9 of the present invention is prepared according to the above preparation route, wherein the reaction conditions of the steps are as follows:

i. 1-hydroxybenzotriazole, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N, N-diisopropylethylamine, N, N-dimethylformamide, room temperature.

ii. Sodium hydroxide, methanol, water, 55 degree

iii 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, N, N-diisopropylethylamine, N, N-dimethylformamide, 0 deg.C

iv, sodium borohydride, methanol, room temperature

v. dessimutan oxidant, ultra-dry dichloromethane, room temperature

The specific synthesis steps are as follows:

intermediate 25: preparation of methyl (1R, 2S, 5S) -6, 6-dimethyl 3- (quinoline-2-carbonyl) -3-azabicyclo [3.1.0] hexane-2-carboxylic acid

Raw material 23 (1.0g, 11.6mmol of quinoline 2-carboxylic acid), 1-hydroxybenzotriazole (2.03g, 15.08mmoL), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (4.43g, 23.3 mmol) and 30mL of N, N-dimethylformamide were placed in a round-bottomed flask and stirred at room temperature for 0.5 hour, 2.5mL of N, N-diisopropylethylamine was added, 0.59g of intermediate 24 was further added, reaction was carried out for 8 hours, the solvent was distilled off under reduced pressure, extraction was carried out with a dichloromethane, an ammonium chloride solution and a sodium hydrogen carbonate solution, washing was carried out with water and a saturated sodium chloride solution, drying was carried out with sodium sulfate, suction filtration was carried out, and an organic phase column was chromatographed to obtain a white solid. The yield thereof was found to be 85%. ¹ H NMR (400MHz, DMSO) delta 8.12-8.03 (m, 1H), 8.00-7.90 (m, 2H), 7.66-7.48 (m, 3H), 4.54 (s, 1H), 4.03 (q, J =7.1Hz, 1H), 3.75 (d, J =6.7Hz, 3H), 3.47 (d, J =5.4Hz 1H), 3.39 (d, J =5.7Hz, 1H), 1.99 (t, J =6.2Hz, 1H), 1.54 (t, J =6.8Hz, 1H), 1.03 (s, 3H), 0.97 (s, 3H). MS (ESI, positive ion) m/z: 325.04.87M + H] ⁺ 。

Intermediate 26: preparation of (1R, 2S, 5S) -6, 6-dimethyl 3- (quinoline-2-carbonyl) -3-azabicyclo [3.1.0] hexane-2-carboxylic acid

Dissolving the intermediate 25 in 20mL of tetrahydrofuran, slowly adding 10mL of 2mol/L sodium hydroxide solution, gradually heating the reaction solution to 55 ℃, stirring for 3 hours, stopping, cooling to normal temperature, concentrating the reaction solution, adding water to adjust the pH value to be subacidity, separating out a white solid, and performing suction filtration to obtain an intermediate 5 which is used for next reaction without further purification

Intermediate 28: preparation of methyl ((1R, 5S) -6, 6-dimethyl-3- (quinoline-2-carbonyl) -3-azabicyclo [3.1.0] hexane-2-carbonyl) -L-phenylalanine

Adding 1.0g of the intermediate 26 and 1.93g of 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate into 20mL of N, N-dimethylformamide, stirring at 0 ℃ for 0.5 hour, adding 2.0mL of N, N-diisopropylethylamine, adding 0.89g of the intermediate, reacting at 0 ℃ under the protection of argon for 12 hours, adding 4 times of water, and extracting with ethyl acetate for three times. Mixing organic phases, extracting with ammonium chloride solution and sodium bicarbonate solution, washing with water and saturated sodium chloride solution, drying with sodium sulfate, vacuum filtering, and subjecting the organic phase to column chromatography to obtain white solid. The yield thereof was found to be 65%. ¹ H NMR(400MHz,DMSO)δ8.53(d,J＝7.5Hz,1H),8.04(d,J＝6.4Hz,1H),7.99(d,J＝6.4Hz,1H),7.94(d,J＝3.7Hz,1H),7.83(d,J＝8.5Hz,1H),7.61–7.57(m,1H),7.33(dd,J＝8.4,1.5Hz,1H),7.28(s,2H),7.23–7.18(m,1H),7.13(d,J＝1.6Hz,1H),6.95(d,J＝2.5Hz,1H),4.55–4.44(m,1H),3.95(d,J＝5.2Hz,1H),3.81(t,J＝11.3Hz,1H),3.61(s,3H),3.05(d,J＝13.8,7.5Hz,1H),2.84(dd,J＝13.8,5.6Hz,1H),2.82(d,J＝5.6Hz,1H),1.42–1.37(m,1H),1.36–1.32(m,1H),0.96(s,3H),0.91(s,3H).

Intermediate 29: preparation of (1R, 5S) -N- ((S) -1-hydroxy-3-phenyn-2-yl) -6, 6-dimethyl-3- (quinoline-2-carbonyl) -3-azabicyclo [3.1.0] hexane-2-carboxamide

500mg of intermediate 28 (((1R, 5S) -6, 6-dimethyl-3- (quinoline-2-carbonyl) -3-azabicyclo [3.1.0] hexane-2-carbonyl) -L-phenylalanine) was dissolved in 30mL of dry methanol, and sodium borohydride was added thereto at room temperature, and after stirring for 3 hours, water was added thereto for quenching, methanol was dried, the aqueous phase (50 mL. Times.3) was extracted with ethyl acetate, the organic phase was collected and dried over sodium sulfate, and after suction filtration, the organic phase was dried to give intermediate 29 in a yield of 80%.

Compound 9: preparation of (1R, 5S) -6, 6-dimethyl-N- ((S) -1-oxo-3-phenylprop-2-yl) -3- (quinoline-2-carbonyl) -3-azabicyclo [3.1.0] hexane-2-carboxamide

Intermediate 29 (1R, 5S) -N- ((S) -1-hydroxy-3-phen-2-yl) -6, 6-dimethyl-3- (quinoline-2-carbonyl) -3-azabicyclo [3.1.0]Dissolving hexane-2-formamide 200mg in10 mL of dry dichloromethane, adding desmartin oxidant under the condition of stirring at normal temperature, monitoring the reaction by TLC to be finished, filtering to remove the oxidant, and performing column chromatography on filtrate to obtain a compound 9 with the yield of 65%. ¹ H NMR(400MHz,DMSO)δ9.55(s,1H),9.06(s,1H),8.56(d,J＝7.3Hz,1H),8.44(dd,J＝8.5,2.4Hz,1H),7.86(d,J＝8.5Hz,1H),7.76(m,1H),7.32–7.21(m,5H),7.14(d,J＝2.9Hz,,1H),7.08(d,J＝5.7Hz,1H),5.33(s,1H),4.44(d,J＝5.6Hz,1H),4.33(m,2H),4.13(m,2H),1.82(m,1H),1.50(m,1H),1.02(s,3H),0.87(s,3H)。

Example 4: preparation of Compound 14

Compound 14 of the present invention is prepared according to the above preparation route, wherein the reaction conditions of the steps are as follows:

i. trifluoroacetic acid, dichloromethane, 25 deg.C

ii. 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate, N, N-diisopropylethylamine and N, N-dimethylformamide at room temperature.

iii, sodium hydroxide, methanol, water, 55 degree

iv, 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, N, N-diisopropylethylamine, N, N-dimethylformamide, 0 deg.C

v, sodium borohydride, methanol, room temperature

vi, dessimutan oxidizer, ultra-dry dichloromethane, room temperature.

The specific synthesis steps are as follows:

intermediate 30: preparation of methyl (S) -2-amino-3- ((S) -2-carbonyl-3-yl) propionate trifluoroacetate

(S) -methyl 2- (tert-butoxycarbonylamino) -3- ((S) -2-carbonylpyrrolidin-3-yl) propionate (intermediate 14,2.5 g) was dissolved in 30mL of dichloromethane, followed by addition of 20mL of trifluoroacetic acid, stirring at room temperature for 14 hours, and direct spin-drying to give a crude product which was used directly in the next reaction.

Intermediate 32: preparation of methyl (1R, 2S, 5S) -6, 6-dimethyl-3- (2- (4- (trifluoromethoxy) phenoxy) acetyl) -3-azabicyclo [3.1.0] hexane-2-carboxylic acid

2- (4- (trifluoromethoxy) phenoxy) acetic acid (commercially available intermediate 31,0.24g, 1.0mmol), 2- (7-oxybenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (0.49g, 1.2mmol) and N, N-diisopropylethylamine (494. Mu.L, 3 mmol) were dissolved in N, N-dimethylformamide and (1R, 2S, 5S) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxylic acid methyl ester hydrochloride (commercially available intermediate 24,0.21g, 1.0mmol), argon-protected reaction at room temperature for 12 hours, 4 times volume of water was added for reaction, extraction was performed three times with dichloromethane, the combined organic phases were washed with a saturated ammonium chloride solution, a saturated sodium carbonate solution, dried over anhydrous sodium sulfate and filtered, and silica gel column chromatography (petroleum ether/ethyl acetate = 1) gave intermediate 32 (0.34g, 88%) as a white solid. ¹ H NMR(400MHz,MeOD)δ7.19(d,J＝8.9Hz,2H),7.00(d,J＝8.7Hz,2H),4.80-4.71(m,2H),4.78–4.72(m,1H)3.89-3.72(m,1H),3.73(s,3H),3.67-3.60(m,1H),1.61-1.55(m,1H),1.49(d,J＝7.4Hz,1H),1.08(s,3H),0.97(s,3H).ESI-MS(m/z):389.08(M+H) ⁺ .

Intermediate 33: preparation of (1R, 2S, 5S) -6, 6-dimethyl-3- (2- (4- (trifluoromethoxy) phenoxy) acetyl) -3-azabicyclo [3.1.0] hexane-2-carboxylic acid

Methyl (1R, 2S, 5S) -6, 6-dimethyl-3- (2- (4- (trifluoromethoxy) phenoxy) acetyl) -3-azabicyclo [3.1.0] hexane-2-carboxylic acid (intermediate 32, 200 mg) was dissolved in 30mL of methanol, 20mL of 2M NaOH solution was added, the mixture was stirred at room temperature for 2.5 hours, after the TLC monitoring reaction was completed, methanol was dried, the pH was adjusted to weak acidity with hydrochloric acid, dichloromethane was extracted three times, and the combined organic phases were dried over anhydrous sodium sulfate and then dried to obtain a crude product, which was directly subjected to the next reaction.

Intermediate 34: preparation of methyl (1R, 2S, 5S) -6, 6-dimethyl-3- (2- (4- (trifluoromethoxy) phenoxy) acetyl) -3-azabicyclo [3.1.0] hexane- -2-amide) -3- ((S) -2-oxo-3-yl) propionate

To (1R, 2S, 5S) -6, 6-dimethyl-3- (2- (4- (trifluoromethoxy) phenoxy) acetyl) -3-azabicyclo [3.1.0] at 0 deg.C]Ultra-dry D of Hexane-2-Carboxylic acid (intermediate 33,0.45g, 1.2mmol)2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (0.61g, 1.6 mmol) was added to the MF solution, and after stirring for 30 minutes, N.N-diisopropylethylamine (0.59mL, 3.6 mmol) was added, and then the crude intermediate 14 (0.27g, 1.45mmol) was added to the reaction system. The reaction was stirred for 12 hours at 0 ℃ under argon protection. After TLC monitoring the reaction was complete, 4 volumes of water were added, extracted three times with ethyl acetate, the combined organic phases were washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and filtered, and column chromatography was performed with a pad column (ethyl acetate/methanol = 10) to afford a white solid as intermediate 34. ¹ H NMR(400MHz,MeOD)δ7.17(d,J＝8.0Hz,2H),6.97(d,J＝8.9Hz,2H),4.80–4.67(m,2H),4.55(d,J＝11.8Hz,1H),3.94-3.83(m,1H),3.72(s,3H),3.68–3.57(m,1H),3.23–3.12(m,1H),3.11–3.00(m,2H),2.58(d,J＝8.8Hz,1H),2.26–2.04(m,2H),1.73–1.40(m,4H),1.10(s,3H),0.92(s,3H).ESI-MS(m/z):542.13(M+H) ⁺ .

Intermediate 35: preparation of (1R, 2S, 5S) -N- ((S) -1-hydroxy-3- ((S) -2-oxo-3-yl) propano-N-2-yl) -3- (2- (4- (trifluoromethoxy) phenoxy) acetyl) -3-azabicyclo [3.1.0] hexane-2-carboxamide

Methyl (1R, 2S, 5S) -6, 6-dimethyl-3- (2- (4- (trifluoromethoxy) phenoxy) acetyl) -3-azabicyclo [3.1.0] hexane- -2-amide) -3- ((S) -2-carbonyl-3-yl) propionate (intermediate 34.56mg, 1.1mmol) was added to 50mL, sodium borohydride (0.14g, 8.8mmol) was added in portions at low temperature, after stirring for 2 hours at normal temperature, water was added for quenching, methanol was spin-dried, the remaining aqueous phase was extracted with ethyl acetate (50 mL. Times.3), the organic phases were combined, dried over anhydrous sodium sulfate, and spin-dried by filtration to obtain a white solid as a crude product, which was directly used in the next reaction.

Compound 14: preparation of (1R, 2S, 5S) -6, 6-dimethyl-N- ((S) -1-aldehyde-3- ((S) -2-oxo-3-yl) propano-2-yl) -3- (2- (4- (trifluoromethoxy) phenoxy) acetyl) -3-azabicyclo [3.1.0] hexane-2-carboxamide

(1R, 2S, 5S) -N- ((S) -1-hydroxy-3- ((S) -2-carbonyl-3-yl) propanoic acid ester-2-yl) -3- (2- (4- (trifluoromethoxy) phenoxy) acetyl) -3-azabicyclo [ 3.1.0)]Hexane-2-carboxamide (intermediate 36, (0.38g, 0.75mmol) was dissolved in ultra dry bisTo methyl chloride, dessimutan oxidant (0.95mg, 0.79mmol) was then added in portions, the reaction was carried out at room temperature for 3.5 hours, TLC monitored for completion of the reaction, the reaction system was filtered, the organic phase was washed with sodium thiosulfate solution and saturated sodium bicarbonate solution, and after concentration, compound 14 (0.28g, 45%) as a white solid sol was isolated with preparative chromatography system (acetonitrile/water = 30. ¹ H NMR(400MHz,MeOD)δ7.24–7.13(m,2H),7.04–6.92(m,2H),4.49(d,J＝9.1Hz,1H),4.37-4.30(m,1H),4.06–3.89(m,1H),3.65-3.49(m,1H),3.11–2.98(m,1H),2.55(d,J＝9.5Hz,1H),2.29–2.10(m,1H),2.06–1.99(m,1H),1.72–1.44(m,4H),1.13(s,3H),1.00(s,3H). ¹³ C NMR(101MHz,MeOD)δ181.60,172.58,167.18,156.90,142.99,121.99,115.47(d,J＝7.7Hz),66.06,61.05,60.14,51.26,46.00,39.94,37.75,30.87(d,J＝7.8Hz),29.88(d,J＝18.5Hz),27.66(d,J＝17.9Hz),25.03,19.04,11.63.HRMS(m/z):calculated for C ₂₄ H ₂₈ F ₃ N ₃ O ₆ ⁺ [M+H] ⁺ 512.1964；found,512.2137.

Example 5: preparation of Compound 15

Compound 15 of the present invention was prepared according to the above preparation route, wherein the reaction conditions in the steps of the route are as follows:

i. 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, N, N-diisopropylethylamine, N, N-dimethylformamide, 0 deg.C

ii. Sodium borohydride, methanol, room temperature

iii, dessimutane oxidizer, ultra-dry dichloromethane, room temperature.

The specific synthesis steps are as follows:

intermediate 36: preparation of methyl (S) -2- ((1S, 3aR, 6aS) -2- (2, 4-dichlorophenoxy) acetyl) octahydrocyclopenta [ c ] pyrrole-1-carboxamide) -3- ((S) -2-oxo-3-yl) propionate

First, 2- (7-benzotriazol oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (0.099g, 0.26mmol) was added to (1S, 3aR,6 aS) -2- (2, 4-Dichlorophenoxy) acetyl) octahydrocyclopenta [ c]Pyrrole-1-carboxylic acid (intermediate 23,0.071g, 0.20mmol) in an ultra-dry N, N-dimethylformamide solution, the reaction system was stirred for 30 minutes, N-diisopropylethylamine (100 μ L,0.60 mmol), methyl (S) -2-amino-3- ((S) -2-carbonyl-3-yl) propionate trifluoroacetate (intermediate 30,0.060g, 0.32mmol) were sequentially added to the reaction system, the reaction was reacted at 0 ℃ under an argon protection condition for 12 hours, TLC monitored for the completion of the reaction, 4 times by volume of water was added, extraction was performed three times with ethyl acetate, the combined organic phases were washed with a saturated ammonium chloride solution, a saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and filtered, and column chromatography was performed (ethyl acetate/methanol = 10) to obtain intermediate 36 (0.063g, 59%). ¹ H NMR(400MHz,MeOD)δ7.40(d,J＝2.5Hz,1H),7.26–7.19(m,1H),6.95(d,J＝8.9Hz,1H),4.79-4.71(m,2H),4.54(d,J＝3.7Hz,1H),4.25(t,J＝4.3Hz,1H),3.72(s,3H),3.54–3.46(m,2H),3.21–3.12(m,1H),3.10–2.99(m,1H),2.86-2.70(m,2H),2.04–1.97(m,2H),1.96-1-85(m,3H),1.83–1.46(m,6H).ESI-MS(m/z):526.03(M+H) ⁺ .

Intermediate 37: preparation of (1S, 3aR,6 aS) -2- (2, 4-dichlorophenoxy) acetyl) -N- ((S) -1-hydroxy-3- ((S) -2-oxo-3-yl) propanoate-2-yl) octahydrocyclopenta [ c ] pyrrole-1-carboxamide

Methyl (S) -2- ((1S, 3aR,6 aS) -2- (2, 4-dichlorophenoxy) acetyl) octahydrocyclopenteno [ c ] pyrrole-1-formamide) -3- ((S) -2-carbonyl-3-yl) propionate (intermediate 36,1,00g,2.0 mmol) is dissolved in anhydrous methanol, then sodium borohydride (0.6 g, 16mmol) is added in batches under the condition of 0 ℃, then the temperature is raised to room temperature, stirring is continued for 2 hours, TLC detection reaction is finished, water is added for quenching, methanol is dried in a spinning mode, the residual aqueous phase is extracted by ethyl acetate (50 mL multiplied by 3), organic phases are combined and dried by anhydrous sodium sulfate, and filtration is carried out to obtain a white solid which is a crude product and is directly used for next reaction.

Compound 15: preparation of (1S, 3aR, 6aS) -2- (2, 4-dichlorophenoxy) acetyl) -N- ((S) -1-aldehyde-3- ((S) -2-carbonyl-3-yl) propanoate-2-yl) octahydrocyclopenta [ c ] pyrrole-1-carboxamide

In (1S, 3aR, 6aS) -2- (2, 4-dichlorophenoxy) acetyl) -N- ((S) -1-hydroxy-3- ((S) -2-carbonyl-3-yl) propanoate-2-yl) octahydrocyclopenta [ c]Pyrrole-1-carboxamide (intermediate 37,0.50g, 1.0mmol) in ultra-dry dichloromethane, was added slowly in portions to dessimutane oxidant (0.55g, 1.3mmol) and reacted at room temperature for 3.5 hours, TLC monitored the reaction end, the reaction system was filtered, the organic phase was washed with sodium thiosulfate solution and saturated sodium bicarbonate solution, and after concentration, compound 30 (0.21g, 42%) was obtained as a white solid using preparative chromatography (acetonitrile/water = 45. ¹ H NMR(400MHz,MeOD)δ7.42(t,J＝4.3Hz,1H),7.28–7.17(m,1H),7.03–6.90(m,1H),4.47(dd,J＝9.6,6.1Hz,1H),4.26(t,J＝5.8Hz,1H),4.03–3.86(m,2H),3.51(dd,J＝10.4,4.0Hz,1H),3.15(t,J＝8.4Hz,1H),3.04–2.82(m,2H),2.75–2.50(m,2H),2.18(dd,J＝13.2,7.0Hz,1H),2.18(dd,J＝13.2,7.0Hz,1H),2.08–1.77(m,5H),1.76–1.43(m,5H). ¹³ C NMR(101MHz,MeOD)δ181.64,173.39,167.10(d,J＝2.9Hz),152.81,129.30,127.35,125.73,123.12,114.81,66.81,60.14,53.90(d,J＝35.6Hz),52.20,51.20(d,J＝18.6Hz),43.34,40.00,37.73,31.69(d,J＝4.2Hz),31.14(d,J＝2.6Hz),29.62(d,J＝24.1Hz),27.67,24.69,19.48,13.09.HRMS(m/z):calculated for C ₂₃ H ₂₇ Cl ₂ N ₃ O ₅ ⁺ [M+H] ⁺ 496.1361；found,496.0842.

Example 6: preparation of Compound 42

Compound 42 of the present invention is prepared according to the above preparation route, wherein the reaction conditions of the steps are as follows:

i、LDA,ClCH ₂ I,THF

ii. HCl dioxane solution

iii, 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate, N, N-diisopropylethylamine, N, N-dimethylformamide, 0 ℃.

The specific synthesis steps are as follows:

intermediate 38: preparation of tert-butyl ((S) -4-chloro-3-oxo-1- ((S) -2-carbonyl-3-yl) butan-2-yl) carbamate

A dry three-necked flask was selected, and argon gas protection and a thermometer were prepared, respectively, and (S) -methyl 2- (tert-butoxycarbonylamino) -3- ((S) -2-carbonylpyrrolidin-3-yl) propionate (intermediate 16,5g, 17.5mmol), tetrahydrofuran (50 mL), chloroiodomethane (5mL, 68mmol) were added and stirred at-77 ℃ lithium diisopropylamide (70mL, 105mmol) was further added dropwise. After the addition was completed, the reaction was further carried out for 2 hours, and then quenched by adding acetic acid and tetrahydrofuran at a low temperature, and the resulting black suspension was further stirred for 10 minutes while warming to room temperature. The reaction was further diluted with ethyl acetate, washed with water, a saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, sample-mixed and column-chromatographed to give a pale yellow solid as intermediate 38. ¹ H NMR(400MHz,DMSO-d ₆ )δ7.89(s,1H),7.72(d,J＝7.5Hz,1H),4.72-4.94(m,2H),4.35(m,1H),3.26-3.40(m,2H),2.45(m,1H),2.32-2.42(m,1H),2.00-2.14(m,1H),1.79-1.99(m,2H),1.61(s,9H)。

Intermediate 39: preparation of (S) -3- ((S) -2-amino-4-chloro-3-oxobutyl) pyrrolidin-2-one hydrochloride

After 250mg of t-butyl ((S) -4-chloro-3-oxo-1- ((S) -2-carbonyl-3-yl) butan-2-yl) carbamate (intermediate 38) was added to 20mL of dioxane, 20mL of hydrogen chloride dioxane solution was added, and the mixture was stirred at room temperature for 4 hours, TLC detected completion of the reaction, and the reaction solution was spin-dried to obtain a crude product for the next reaction.

Compound 42: preparation of (1S, 3aR, 6aS) -N- ((S) -4-chloro-3-oxo-1- ((S) -2-oxo-3-yl) butan-2-yl) -2- (2, 4-dichlorophenoxy) acetyl) octahydro-cyclopenta [ c ] pyrrole-1-carboxamide

2- (7-Oxotobenzotriazol) -N, N, N ', N' -Tetramethyluronium hexafluorophosphate (1g, 2.6 mmol) was added to (1S, 3aR,6 aS) -2- (2, 4-dichlorophenoxy) acetyl) octahydrocyclopenta [ c ] c]In an ultra-dry N, N-dimethylformamide solution of pyrrole-1-carboxylic acid (intermediate 23,0.71g, 2.0mmol), the reaction system was stirred for 30 minutes, N, N-diisopropylethylamine (1mL, 6.0mmol), (S) -3- ((S) -2-amino-4-chloro-3-oxobutyl) pyrrolidin-2-one hydrochloride (intermediate 39,0.652g, 3.2mmol) was added to the reaction system in this order, the reaction was reacted at 0 ℃ under argon protection for 16 hours, and TLC was used to monitor the reaction junctionThe mixture was extracted three times with ethyl acetate by adding 3 volumes of water, and the combined organic phases were washed with a saturated ammonium chloride solution and a saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and filtered to separate compound 42 from the preparative purification system (acetonitrile/water = 30). ¹ H NMR(400MHz,MeOD)δ7.40(s,1H),7.20(dd,J＝8.7,2.4Hz,1H),7.05–6.91(m,1H),5.00–4.89(m,1H),4.79(d,J＝16.1Hz,1H),4.69–4.35(m,2H),4.25(m,1H),3.99–3.85(m,1H),3.58–3.45(m,1H),3.25–3.03(m,1H),2.93–2.77(m,1H),2.76–2.50(m,2H),2.45–2.24(m,1H),2.18(d,J＝10.9Hz,1H),2.11–1.47(m,9H),1.39–1.27(m,1H).ESI-MS(m/z):544.07(M+H) ⁺ .

Example 7: preparation of Compound 50

Compound 50 of the present invention was prepared according to the above preparation route, wherein the reaction conditions in the steps of the route are as follows:

i. tert-butyl isobutyronitrile, acetic acid and ultra-dry dichloromethane;

ii. 1M sodium hydroxide solution, methanol;

iii, dessimutan oxidizer, ultra-dry dichloromethane.

The specific synthesis steps are as follows:

intermediate 40: preparation of (3S) -1- (tert-butylamino) -3- ((1S, 3aR,6 aS) -2- (2, 4-dichloro) acetyl) octahydrocyclopenta [ c ] pyrrole-1-carboxamide) -1-oxo-4- ((S) -2-oxo-3-yl) butan-2-ylcarboxylic acid

First, compound 15 (0.40 mmol) was dissolved in ultra-dry dichloromethane, followed by the addition of acetic acid (0.028g, 0.47mmol), tert-butylisonitrile (0.43 mmol) in that order. The reaction was stirred at room temperature for 24 hours, and chromatographed on a reduced pressure distillation column (dichloromethane/methanol = 15) to give intermediate 40. ¹ H NMR(400MHz,DMSO-d ₆ )δ7.87(d,J＝12.1Hz,1H),7.46(s,1H),7.44(d,J＝1.4Hz,1H),7.37(t,J＝4.6Hz,1H),7.27(dd,J＝7.5,1.5Hz,1H),7.11(d,J＝7.5Hz,1H),5.09(d,J＝7.1Hz,1H),4.82(s,2H),4.36–4.28(m,2H),3.64(ddd,J＝59.5,12.4,7.0Hz,2H),3.22(td,J＝7.1,4.6Hz,2H),2.67–2.44(m,3H),2.09(s,3H),1.99–1.51(m,10H),1.27(s,9H)。

Intermediate 41: preparation of (1S, 3aR,6 aS) -N- ((2S) -4- (tert-butylamino) -3-hydroxy-4-oxo-1- ((S) -2-oxo-3-yl) butan-2-yl) -2- (2, 4-dichloro) acetyl) octahydrocyclopenta [ c ] pyrrole-1-carboxamide

1M sodium hydroxide solution was added (0.5 mL) to a solution of intermediate 40 (0.164 mmol) in methanol, the reaction was stirred at ambient temperature for 2 hours, the pH was adjusted to neutral with 1M hydrochloric acid, the reaction solution was spin dried, the residue was dissolved in dichloromethane, and the reaction solution was spin dried after extraction with water to give crude product 41 which was used directly in the next reaction.

Compound 50: preparation of (1S, 3aR,6 aS) -N- ((S) -4- (tert-butylamino) -3, 4-oxo-1- ((S) -2-oxo-3-yl) butan-2-yl) -2- (2-2, 4-dichloro) acetyl) octahydrocyclopenta [ c ] pyrrole-1-carboxamide

To the ultra-dry dichloromethane solution of intermediate 41, dessimutan oxidizer was slowly added in portions, the reaction was carried out at room temperature for 4 hours, TLC monitored for completion of the reaction, the reaction system was filtered, the organic phase was washed with sodium thiosulfate solution and saturated sodium bicarbonate solution, and after concentration, compound 50 was obtained as a white solid using a preparative chromatography system (acetonitrile/water = 50. ¹ H NMR(400MHz,MEOD)δ7.42(d,J＝1.4Hz,1H),7.26(dd,J＝7.5,1.5Hz,1H),7.12(d,J＝7.5Hz,1H),4.84(s,2H),4.67(dt,J＝11.9,7.0Hz,1H),4.36(dd,J＝7.0,0.7Hz,1H),3.74–3.57(m,2H),3.23–3.13(m,2H),2.70–2.46(m,3H),2.15–1.54(m,10H),1.43(s,9H).ESI-MS(m/z):595.07(M+H) ⁺ 。

The remaining compounds of the present invention shown in Table 1 were obtained by changing the starting materials according to the preparation routes in examples 1 to 7.

TABLE 1 Structure and characterization data for the Compounds of the invention

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

The pharmacological effects of the compounds of the present invention are demonstrated by the following experimental examples.

Experimental example 1: compound pair M of the present invention ^pro Assay for level of inhibition of enzyme Activity

(1) Experimental methods

Recombinant SARS-CoV-2M ^pro (final concentration 750 nM) was mixed with serial dilutions of each compound in 25. Mu.L assay buffer (20 mM Tris-HCl, pH 7.5, 150mM NaCl,1mM EDTA,2mM DTT) and incubated for 10 min. The reaction was initiated by the addition of 25. Mu.L of fluorogenic substrate (MCA-AVLQ ↓ SGFR-Lys (Dnp) -Lys-NH 2) at a final concentration of 20. Mu.M, and the fluorescence signal at 320nm (excitation)/405 nm (emission) was measured with a microplate reader. Vmax of reactions with different concentrations of compound added and Vmax of reactions with DMSO added were calculated and used to generate IC ₅₀ Curve line. anti-SARS-CoV-2M measurements at 9 concentrations and 3 independent replicates for each compound ^pro semi-Inhibitory Concentration (IC) ₅₀ ) The value is obtained. All experimental data were analyzed using GraphPad Prism software.

(2) Results of the experiment

TABLE 2 Compound vs SARS-COV-2M ^pro Enzyme activity inhibitory effect of

/>

As can be seen from Table 2 and FIGS. 1, 2 and 3, the compounds of the present invention are effective in inhibiting SARS-CoV-2M ^pro Can be used for preparing SARS-CoV-2M ^pro Inhibitors, the preparation of medicaments against novel coronaviruses and the preparation of medicaments for the prophylaxis and/or treatment of novel coronaviruses.

Experimental example 2: inhibition experiment of SARS-COV-2 infection Vero E6 cell to cell death

(1) Experimental methods

The antiviral activity of the compound is preliminarily evaluated by detecting the inhibition effect of the compound on cell death caused by SARS-COV-2 infected Vero E6 cells. The specific experimental scheme is as follows: vero E6 cells were grown at a cell density of 2X 10 ⁴ Cells/well, 100. Mu.L/well seeded in 96-well plates, 5% CO at 37% ₂ Incubate overnight in an incubator. The following day, 100. Mu.L of drug and 100. Mu.L of virus diluent (MOI = 1) were added simultaneously to each well, a drug-free positive control and a virus-free negative control were set, 37 ℃,5% CO ₂ Culturing for 72h, detecting cell survival rate with CCK-8 kit, and calculating drug inhibition rate on virus replication and half-onset concentration (EC) ₅₀ ) Values, 3 independent replicates were set for all experiments and all experimental data were analyzed using GraphPad Prism software.

(2) Results of the experiment

TABLE 3 inhibitory Activity of the Compounds of the present invention against cell death caused by SARS-COV-2 infection of Vero E6 cells

/>

/>

Note: NT stands for untested cell Activity

As can be seen from Table 3, the compound of the invention can effectively inhibit cell death caused by SARS-COV-2 infecting Vero E6 cells, which shows that the compound of the invention can effectively inhibit the replication of SARS-COV-2 virus in cells.

Experimental example 3: toxicity test of Compounds on Vero E6 cells

(1) Experimental methods

Cytotoxicity assessment of compounds was performed using Vero E6 cells. The specific experimental scheme is as follows: vero E6 cells were grown at a cell density of 2X 10 ⁴ Cells/well, 100. Mu.L/well were seeded in 96-well plates and incubated overnight in a 5% CO2 incubator at 37 ℃. The next day, 200 μ L of drug-containing medium was added to each well, and the compound was diluted in 5-fold gradients at 200 μ M for 6 gradients, 3 replicate wells were set for each concentration, and a negative control and blank control without drug were set for each experiment. After 72h of drug treatment, using CCK-8 kit to detect cell viability, calculating toxicity and half-cell toxicity concentration (CC) of compound to Vero E6 cell ₅₀ ) The value is obtained. All experimental data were analyzed using GraphPad Prism software.

(2) Results of the experiment

TABLE 4 toxicity of the Compounds of the invention on Vero E6 cells

/>

/>

As can be seen from Table 4, the compounds of the invention have very low toxicity to Vero E6 cells.

Experimental example 4: inhibition experiment of SARS-COV-2 replication in human alveolar epithelial cells

(1) Experimental method

For the RT-qPCR method, human alveolar epithelial cells were cultured at 8X 10 ⁵ The density of individual cells/well was seeded into 48-well plates (200. Mu.L/well) and grown overnight. Cells were then treated with virus infection (MOI = 0.01) and different concentrations of compound. After 1 hour incubation at 37 ℃, the medium containing the virus-drug mixture was removed and replaced with fresh medium containing the compound. After an additional 48 hours of incubation, cells were harvestedExtracting virus RNA from the supernatant, carrying out RT-qPCR quantitative analysis on the virus RNA and calculating the inhibition rate and EC of the drug on virus replication ₅₀ The value is obtained. EC (EC) ₅₀ Values were calculated using a dose response model in GraphPad Prism 8.0 software, with 2 independent replicates in the experimental setup.

(2) Results of the experiment

As shown in FIG. 4, compounds 14, 15, 26, 43, 44 and 45 all showed nanomolar inhibitory activity against SARS-COV-2 replication in human alveolar epithelial cells, superior to the reported SARS-COV-2M with the highest activity ^pro Antiviral activity of inhibitors 11b (Dai et al, 2020, science.368 (6497): 1331-1335) and GC376 (Ma et al, 2020, cell Res.30 (8): 678-692) under the same test conditions (11b, EC) ₅₀ ＝23.6nM；GC376，EC ₅₀ ＝151.3nM)。

Experimental example 5: evaluation of Compounds against SARS-COV-2 Activity by plaque assay (Vero E6 cells)

(1) Experimental methods

Compounds 3 and 39 were evaluated for anti-SARS-COV-2 activity in Vero E6 cells using the plaque method. Vero E6 at 1.0X 10 per well ⁵ One was inoculated into a 24-well cell culture plate and cultured overnight at 37 ℃ for use. After addition of the serially diluted drug, SARS-CoV-2 was added to infect the cells with an MOI of about 0.002. After culturing in a 37 ℃ cell culture box for 1 hour, the drug-containing infection supernatant is removed, PBS is washed once, 0.5mL of sodium carboxymethylcellulose containing drugs with different concentrations and the final concentration of the sodium carboxymethylcellulose is 0.9 percent are added, and the mixture is cultured in the 37 ℃ cell culture box for 72 hours. After fixation with 20% formaldehyde for 2 hours, 0.5% crystal violet was added and stained for 20 minutes, the photographs were air-dried, the size of the plaques was observed and the number of plaques was recorded. Blank control wells (normal cells), virus control wells, and positive drug control wells were set for the experiment.

Calculating the formula: inhibition (%) = (number of plaques in virus control well-number of plaques in sample well)/number of plaques in virus control well × 100

The activity and inhibition rate of the obtained cells were calculated, and EC was calculated using Graphpad Prism8 ₅₀ (half onset concentration) value.

(2) Results of the experiment

TABLE 5 inhibition of SARS-CoV-2 by small molecule compounds

Name of Compound	EC ₅₀ (μM)
		3	0.24
39	1.20
		Remdesivir (Ruidexivir)	0.69

The experimental result is shown in Table 5, the compound of the invention can effectively inhibit SARS-COV-2 infection in Vero E6 cells; in particular the compound 3,EC ₅₀ The activity is 0.2373 mu M, and is better than that of the positive control Reidesciclovir (EC) ₅₀ 0.692. Mu.M).

Experimental example 6: evaluation of in vivo pharmacokinetic Properties of Compounds on rats

(1) Dosing regimens

Male Sprague-Dawley (SD) rats 60, weighing 200-230g, were randomly divided into 3 groups of 3 rats each. The test compounds were administered intragastrically (p.o.), intravenously (i.v.) and intraperitoneally (i.p.) respectively according to the protocol of table 6 below. Fasted for 12h before the experiment, and water was freely drunk. The diets were uniformly fed 2h after dosing.

The gavage, intravenous and intraperitoneal administration solutions were formulated in DMSO/HS15/NaCl (5/3/92, v/v/v). The drugs were administered at the dosing doses shown in table 6, the dosing time was recorded, and approximately 0.20mL of each sample was collected via jugular vein blood collection or other suitable means at the time points set forth above, anticoagulated with heparin sodium, and placed on ice after collection. And the plasma was centrifuged within 1 hour (centrifugation conditions: 6800g,6 minutes, 2-8 ℃ C.). Plasma samples were stored in a-80 ℃ freezer prior to analysis. Grouping and blood sampling time points are shown in table 6, 3 animals per time point.

Table 6 evaluation of in vivo pharmacokinetic Properties of Compounds on rats

(2) Results of the experiment

Table 7 major pharmacokinetic parameters of the compounds

/>

The results are shown in Table 7. The present inventors have conducted pharmacokinetic studies on

compounds

3, 14, 15, 26, 39, 40, 43, 44 and 45. Wherein the oral exposure of compound 3 was 2293h ng/mL, and the bioavailability was 55.1%. Compound 14 had an intraperitoneal exposure of 11581h ng/mL, and a bioavailability of 78.0%; the oral exposure was 1665h ng/mL, bioavailability was 11.2%. Compound 15 was administered by intraperitoneal injection at exposure 12166h ng/mL, with a bioavailability of 62.3%; the oral exposure was 2843h ng/mL, and the bioavailability was 14.6%. Compound 26 had an oral exposure of 842h ng/mL and a bioavailability of 7.2%. The oral exposure of compound 39 was 14586h ng/mL, with a bioavailability of 14.7%. Compound 40 had an oral exposure of 2888h ng/mL and a bioavailability of 22.1%. Compound 43 had an oral exposure of 258h ng/mL and a bioavailability of 4.8%. The oral exposure of compound 44 was 381h ng/mL with a bioavailability of 4.1%. Compound 45 had an oral exposure of 968h ng/mL, and a bioavailability of 5.1%.

The experimental result shows that the compound has good pharmacokinetic property in rats.

Experimental example 7: preliminary evaluation of in vivo safety of Compounds in rats

(1) Experimental methods

Compounds were dissolved in 5% (v/v) DMSO (Sigma-Aldrich), 3% (v/v) HS15 (GLPBIO) and 92% saline. SPF SD rats (age: 7-11 weeks) were 190-220 g female and 200-230g male. The test was performed according to the dosing schedule of table 8 and clinical observations were made for all animals. And at the end of the experiment, samples of the heart, liver, spleen, lung, kidney and administration site were collected. The test results are shown in Table 8.

(2) Results of the experiment

TABLE 8 preliminary evaluation of in vivo safety in rats

The experimental result shows that the compound of the invention has good in-vivo safety to rats.

Experimental example 8: activity study of Compounds against SARS-COV-2 infection in vivo in transgenic mice

(1) Experimental protocol

Humanized angiotensin converting enzyme 2 (ACE 2) transgenic mice (age: 8-10 weeks) were purchased from Jiangsu Jiejiekang biotech Co. (# T037659. Compounds were dissolved in 5% (v/v) DMSO (Sigma-Aldrich), 3% (v/v) HS15 (GLPBIO) and 92% saline. SARS-CoV-2 (stain 107) nasal drip infection and dosing was performed according to the protocol of table 9. All mice were observed and their body weights were monitored daily until sacrificed. Day 1 (1 dpi), 3 (3 dpi) and 5 (5 dpi) post viral infection, lung tissue (n =3, each dpi group) was collected for viral load detection, H & E histopathological analysis, representative inflammatory cytokine and chemokine assays, and inflammatory cell (neutrophil and macrophage) counts.

TABLE 9 Activity Studies of Compounds against SARS-COV-2 infection in transgenic mice

The specific experimental scheme for detecting the lung viral load is as follows: using TRIzol ^TM Reagent (Invitrogen) for extraction of RNA from lung tissue and use

The viral RNA was quantified by a one-step qRT-PCR kit (Toyobo), and the results were expressed as the number of copies of viral RNA per microgram of tissue.

The specific experimental protocol for lung histopathological analysis was: lung tissue was fixed with 4% paraformaldehyde for at least 7 days, embedded in paraffin and cut into 3 μm sections. Sections were stained with hematoxylin and eosin (H & E) and analyzed by light microscopy. Lung injury was assessed according to histological features (thickening of alveolar septa, bleeding, inflammatory cell infiltration, etc.).

Specific embodiments of lung representative inflammatory cytokine and chemokine assays are: using PrimeScript ^TM RT kit (Takara) reverse transcribing RNA extracted from lung into cDNA, followed by

Ex Taq ^TM II (TliRNaseH Plus) (Takara) and ViiA ^TM Gene expression was quantified. Primer sequences for quantifying inflammatory gene expression are shown in table 10.

TABLE 10 determination of primer sequences for representative inflammatory cytokines and chemokines

Specific embodiments for pulmonary determination of the number of inflammatory cells (neutrophils and macrophages) are: mouse lung tissue was fixed in 4% paraformaldehyde for at least 7 days, then paraffin embedded and cut into 4 μm sections according to standard procedures. After deparaffinization in xylene, antigen recovery and blocking, lung sections were incubated with either a rat monoclonal antibody F4/80 (Huabio, 1, 100) or a rabbit polyclonal antibody Ly6G (Servicebio, 1, 300) overnight at 4 ℃, and then reacted with either a horseradish peroxidase (HRP) -conjugated goat-anti-rat secondary antibody or an HRP-conjugated goat-anti-rabbit secondary antibody at room temperature for 1 hour to catalyze Cy 3-tyramine and Cy 5-tyramine according to Tyramide Signal Amplification (TSA) and amplify the staining signal. After staining the nuclei with DAPI, all sections were photographed using a LEICA DMI 4000B microscope (germany) and analyzed by ImageJ software (NIH us) and FlowJo software (BD us). To semi-quantitatively measure macrophage and neutrophil infiltration, 5 arbitrarily selected lung parenchymal areas in each lung section were examined by light microscopy to observe the presence of neutrophils or macrophages. The evaluation was performed in a blind manner. The cumulative score for each animal was expressed as the number of positive fields (%) per 100 fields.

The above experiment was controlled with placebo. The placebo is the same formulation as the test drug formulation, but does not contain a pharmaceutically active ingredient.

(2) Results of the experiment

The lung viral load detection experiment result is shown in figure 5, and the viral load of the lung of a transgenic mouse infected by SARS-COV-2 can be effectively reduced by orally taking the compound 14 and intraperitoneally taking the compound 15.

The lung histopathological analysis experiment result is shown in figure 6, and the oral administration, the intraperitoneal administration of the compound 14 and the intraperitoneal administration of the compound 15 can effectively improve the pathological damage of the lung of a transgenic mouse infected by SARS-COV-2.

The results of the lung representative inflammatory cytokine and chemokine assay are shown in fig. 7, and oral and intraperitoneal administration of compound 14 and compound 15 can effectively reduce the gene expression levels of lung chemokine ligand 10 (CXCL 10) and interferon beta (IFN- β).

The results of the lung assay for the number of inflammatory cells (neutrophils and macrophages) are shown in FIG. 8, and both oral and intraperitoneal administration of compound 14 and compound 15 were effective in reducing the number of Neutrophils (NEU) and Macrophages (MAC) in the lung of transgenic mice infected with SARS-COV-2.

Experimental results show that the compound can effectively resist SARS-COV-2 infection in vivo of transgenic mice.

In conclusion, the invention provides a novel coronavirus main protease inhibitor shown as a formula I, and a preparation method and application thereof. The compound shown in the formula I can effectively inhibit SARS-CoV-2M ^pro Active, can be used for preparing SARS-CoV-2M ^pro Inhibitor for blocking the replication and transcription of SARS-CoV-2 virus in a patient. Application of compound of the invention in preparation of SARS-CoV-2M ^pro The inhibitor, the medicine for resisting SARS-CoV-2 and the medicine for preventing and/or treating the novel coronavirus have very good application prospects.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopically substituted form thereof:

wherein X is O or S;

a ring is selected from unsubstituted or substituted by one or more R ₆ Substituted of the following groups: 5-6 membered saturated heterocyclic group, 5-6 membered unsaturated heterocyclic group, saturated hetero condensed ring group, unsaturated hetero condensed ring group; r ₆ Each independently selected from C _1～6 Alkyl radical, C _1～6 Alkoxy, halogen, hydroxy, cyano, amino, carboxy;

R ₃ is L ₃ M ₀ L ₄ R _3a (ii) a Wherein L is ₃ Selected from the group consisting of _1～4 Alkylene, halogeno C _1～4 Alkylene radical, C _2～4 Alkenylene, halogeno C _2～4 Alkenylene radical, L ₄ Selected from the group consisting of _1～4 Alkylene, halogeno C _1～4 Alkylene radical, M ₀ Selected from among none, O, S, NH, CO, CONH, NHCO，R _3a Is unsubstituted or substituted by one or more R _3b Substituted of the following groups: 5-6 membered aryl, 5-6 membered heteroaryl, unsaturated hetero-condensed ring group, unsaturated condensed ring alkyl; r _3b Each independently is selected from R _3c Substituted or unsubstituted C _1～5 Alkyl radical, by R _3c Substituted or unsubstituted C _1～5 Alkoxy, halogen, by R _3c Substituted or unsubstituted phenyl, NR ₁₄ R ₁₅ Quilt R _3c Substituted or unsubstituted naphthyl, hydroxy; r ₁₄ 、R ₁₅ Each independently selected from hydrogen or C _1～5 Alkyl radical, R _3c Each independently selected from halogen, deuterium, cyano, hydroxyl, amino, carboxyl;

R ₄ selected from the following groups unsubstituted or substituted with one or more substituents: 5-6 membered aryl, 5-6 membered heteroaryl, C _1～5 Alkyl, COOR ₁₀ (ii) a The substituents are each independently selected from = O, hydroxy, nitro, amino, carboxy, halogen, C _1～5 An alkyl group; r ₁₀ Is C _1～5 An alkyl group;

W is selected from the group consisting of _1～4 Alkylene radical, C _2～4 Alkenylene radical, C _2～4 Alkynylene, R ₇ Is selected from C _1～6 An alkyl group; m is selected from among nothing, CO, NH, CONH, NHCO, COO or OCO, L ₀ Selected from among none, C _1～4 Alkylene radical, C _2～4 Alkenylene radical, L ₁ Selected from among none, C _1～4 Alkylene radical, C _2～4 Alkenylene radical, R ^8a Is selected from C _1～5 Alkyl, halogenated C _1～5 Alkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic, 5-6 membered aryl or 5-6 membered heteroaryl.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopically substituted form thereof, wherein: the structure of the compound is shown as formula II, formula III or formula IV:

wherein X is O or S;

n is selected from an integer of 0 to 3, preferably an integer of 0 to 2;

R ₃ is L ₃ M ₀ L ₄ R _3a (ii) a Wherein L is ₃ Selected from the group consisting of _1～4 Alkylene, halogeno C _1～4 Alkylene radical, C _2～3 Alkenylene radical, L ₄ Selected from the group consisting of _1～4 Alkylene, halogeno C _1～4 Alkylene, M ₀ Selected from among none, O, S, NH, CO, CONH, NHCO, R _3a Is unsubstituted or substituted by one or more R _3b Substituted of the following groups: phenyl group,

R _3b Each independently selected from C _1～4 Alkyl, halogen substituted C _1～4 Alkyl, deuterated C _1～4 Alkyl, cyano-substituted C _1～4 Alkyl radical, C _1～4 Alkoxy, halogen substituted C _1～4 Alkoxy, deuterated C _1～4 Alkoxy, cyano-substituted C _1～4 Alkoxy, halogen, phenyl, halogenated phenyl, NR ₁₄ R ₁₅ 、

Hydroxy, R ₁₄ 、R ₁₅ Each independently selected from hydrogen or C _1～4 An alkyl group;

R ₄ selected from unsubstituted or substituted by oneOr the following group substituted with a plurality of substituents: 5-6 membered aryl, 5-6 membered heteroaryl, C _1～5 Alkyl, COOR ₁₀ (ii) a The substituents are each independently selected from = O, hydroxy, nitro, amino, carboxy, halogen, C _1～5 An alkyl group; r ₁₀ Is C _1～5 An alkyl group;

R ₈ selected from hydrogen or

M is selected from among nothing, CO, NH, CONH, NHCO, COO or OCO, L ₀ Selected from among none, C _1～3 Alkylene radical, C _2～4 Alkenylene radical, L ₁ Selected from the group consisting of _1～3 Alkylene radical, C _2～4 Alkenylene radical, R ^8a Is selected from C _1～4 Alkyl, halogenated C _1～4 Alkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclyl, 5-6 membered aryl or 5-6 membered heteroaryl.

3. The compound according to claim 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopically substituted form thereof, wherein:

R ₁ 、R ₂ each independently selected from hydrogen, C _1～4 Alkyl radical, C _1～4 Alkoxy, halogen, hydroxy;

R ₃ is selected from

L ₃ M ₀ L ₄ R _3a ；L ₃ Selected from the group consisting of _1～3 Alkylene, halogeno C _1～3 Alkylene radical, C _2～3 Alkenylene radical, L ₄ Selected from the group consisting of _1～3 Alkylene, halogeno C _1～3 Alkylene, M ₀ Selected from among none, O, NH, CO, CONH, R _3a Is phenyl, substituted by one or more R _3b Substituted phenyl radicals, R _3b Each independently selected from C _1～4 Alkyl, halogenSubstituted C _1～4 Alkyl, deuterated C _1～4 Alkyl, cyano-substituted C _1～4 Alkyl radical, C _1～4 Alkoxy, halogen substituted C _1～4 Alkoxy, deuterated C _1～4 Alkoxy, cyano-substituted C _1～4 Alkoxy, halogen, phenyl, halogenated phenyl, NR ₁₄ R ₁₅ 、/>

R ₄ is selected from

R ₈ selected from hydrogen, CONHR ₁₁ 、L ₂ COOR ₁₂ 、C _1～4 Alkyl, halogenated C _1～4 An alkyl group; r ₁₁ Selected from 3-to 6-membered saturated cycloalkyl, C _1～4 Alkyl, benzyl, or a salt thereof,

4. A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopically substituted form thereof, wherein: the structure of the compound is one of the following structures:

/>

5. a pharmaceutical composition characterized by: the pharmaceutical composition is a preparation prepared by adding pharmaceutically acceptable auxiliary materials into the compound of any one of claims 1 to 4, or pharmaceutically acceptable salt, stereoisomer, optical isomer or isotopic substitution form thereof as an active ingredient.

6. Use of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopically substituted form thereof, for the preparation of a coronavirus proteolytic enzyme inhibitor; preferably, the coronavirus proteolytic enzyme is coronavirus main protease; more preferably, the coronavirus proteolytic enzyme is SARS-COV-2M ^pro 。

7. Use of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopically substituted form thereof, for the manufacture of a medicament for combating coronavirus, preferably the coronavirus is the novel coronavirus SARS-CoV-2.

8. Use of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopically substituted form thereof, for the preparation of a medicament for the prevention and/or treatment of SARS-COV-2M ^pro Use in medicine of related diseases, preferably, the SARS-COV-2M ^pro The related disease is the novel coronavirus COVID-19.

9. Use according to any one of claims 6 to 8, characterized in that: the coronavirus proteolytic enzyme inhibitor, the anti-coronavirus drug or the drug for preventing and/or treating viral pneumonia canInhibition of SARS-COV-2M ^pro And/or is capable of inhibiting SARS-COV-2 infection of cells.